Cholesterol-dependent LXR transcription factor activity represses pronociceptive effects of estrogen in sensory neurons and pain induced by myelin basic protein fragments

Background A bioactive myelin basic protein (MBP) fragment, comprising MBP84-104, is released in sciatic nerve after chronic constriction injury (CCI). Intraneural injection (IN) of MBP84-104 in an intact sciatic nerve is sufficient to induce persistent neuropathic pain-like behavior via robust transcriptional remodeling at the injection site and ipsilateral dorsal root ganglia (DRG) and spinal cord. The sex (female)-specific pronociceptive activity of MBP84-104 associates with sex-specific changes in cholesterol metabolism and activation of estrogen receptor (ESR)1 signaling. Methods In male and female normal and post-CCI rat sciatic nerves, we assessed: (i) cholesterol precursor and metabolite levels by lipidomics; (ii) MBP84-104 interactors by mass spectrometry of MBP84-104 pull-down; and (iii) liver X receptor (LXR)α protein expression by immunoblotting. To test the effect of LXRα stimulation on IN MBP84-104-induced mechanical hypersensitivity, the LXRα expression was confirmed along the segmental neuraxis, in DRG and spinal cord, followed by von Frey testing of the effect of intrathecally administered synthetic LXR agonist, GW3965. In cultured male and female rat DRGs exposed to MBP84-104 and/or estrogen treatments, transcriptional effect of LXR stimulation by GW3965 was assessed on downstream cholesterol transporter Abc, interleukin (IL)-6, and pronociceptive Cacna2d1 gene expression. Results CCI regulated LXRα ligand and receptor levels in nerves of both sexes, with cholesterol precursors, desmosterol and 7-DHC, and oxysterol elevated in females relative to males. MBP84-104 interacted with nuclear receptor coactivator (Ncoa)1, known to activate LXRα, injury-specific in nerves of both sexes. LXR stimulation suppressed ESR1-induced IL-6 and Cacna2d1 expression in cultured DRGs of both sexes and attenuated MBP84-104-induced pain in females. Conclusion The injury-released bioactive MBP fragments induce pronociceptive changes by selective inactivation of nuclear transcription factors, including LXRα. By Ncoa1 sequestration, bioactive MBP fragments render LXRα function to counteract pronociceptive activity of estrogen/ESR1 in sensory neurons. This effect of MBP fragments is prevalent in females due to high circulating estrogen levels in females relative to males. Restoring LXR activity presents a promising therapeutic strategy in management of neuropathic pain induced by bioactive MBP.

MBP 84-104 is a highly evolutionarily conserved motif (identical in humans and rodents, Fig. 1B) comprising an immunodominant T cell epitope implicated in the pathogenesis of autoimmune demyelinating disease, multiple sclerosis (Harauz and Boggs, 2013).Intraneural injection of MBP 84-104 peptide into an intact sciatic nerve  ) is sufficient to initiate a state of mechanical allodynia sustained for up to 4 weeks (Chernov et al., 2018;Hong et al., 2017;Ko et al., 2016;Liu et al., 2012;Remacle et al., 2018).Pronociceptive activity of IN MBP 84-104 is T cell-dependent, as shown using athymic nude rats (Liu et al., 2012), charge-dependent, as shown by mutagenesis of the peptide's H89 site (Chernov et al., 2018), and associates with transcriptional remodeling of lipid energy metabolism (Chernov et al., 2020).

Animals and tissues
Adult female and male Sprague-Dawley rats (Envigo, 8-10-weekold) were housed in plastic cages in temperature-and light-controlled (12-h light/dark cycle) conditions with ad libitum access to food and water.All procedures were conducted during the light cycle.Animals were randomly assigned to the surgical and treatment groups.All animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, the ethical guidelines of the International Association for the Study of Pain, and the experimental protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Diego, the Veterans Affairs San Diego Healthcare System, and the University of Texas MD Anderson Cancer Center.All procedures were designed to minimize animal discomfort and studies were powered to use the fewest possible number of animals.

Animal models
Under isoflurane anesthesia (4% induction, 2% maintenance, Isothesia, Henry Schein), the common sciatic nerve was exposed unilaterally (left side) at the mid-thigh level through a gluteal muscle-splitting incision.Using sterilized micro scissors and Jewelers forceps from Roboz, chronic constriction injury (CCI) was administered by applying three loosely constrictive 5.0 chromic gut ligatures (Ethicon suture 634G) around the sciatic nerve (Bennett and Xie, 1988).Care was taken to produce consistent ligations that constricted no more than one-third of the diameter of the nerve, using a double knot.In a separate group of female rats, a single bolus intraneural (IN) injection of the MBP 84-104  ) or SCR peptides (50 μg in 5 μl phosphate buffered saline (PBS), each) or PBS alone (5 μl) was injected into the sciatic nerve fascicle, using a 33-gauge needle on a Hamilton syringe, as previously described (Chernov et al., 2018;Ko et al., 2016;Liu et al., 2012).In sham operated rats, the left sciatic nerve was exposed, but not ligated.In all groups, muscle was sutured closed using 4.0 perma-hand silk suture (Ethicon 683G) and skin was stapled closed.Aside from isoflurane, no additional analgesics were given.

Tissues collection
For immunoblotting, lipidomics and mass spectrometry analyses, rats were sacrificed by intraperitoneal Euthasol (100-150 mg/ml; Virbac Animal Health).In CCI rats, Ethicon sutures were removed prior to sciatic nerve excision.Tissues were excised, snap-frozen in liquid nitrogen and stored at − 80 • C. For immunostaining, rats were perfused transcardially under deep anesthesia with a saline flush followed by fresh 4% paraformaldehyde (PFA) in 0.2 M phosphate buffer.Sciatic nerve injection, CCI injury (3 mm distal and 3 mm proximal to the ligature location) or anatomically equvalent sites, lumbar (L)4-5 dorsal root ganglia (DRG) and spinal cord lumbar enlargement (L1-L6) tissues were processed for analyses.Tissues contralateral to injury or IN injection, or from sham-operated or naïve animals, were collected for controls.

IT LXR agonist therapy
Intrathecal (IT) catheters were placed and secured, as reported (Malkmus and Yaksh, 2004).In brief, under isoflurane inhalant anesthesia, each animal was given subcutaneous Lactated Ringer's Solution (20 mL/kg) with added carprofen (5 mg/kg).The back of the head and neck was then shaved, and the animal mounted into a stereotaxic unit.The surgical site was prepped with alternating applications of commercially available chlorhexidine gluconate solution and 70% isopropyl alcohol.Using a 15# scalpel blade, a 1 cm skin incision was made along the dorsal midline of the skull, being careful not to incise any of the underlying muscle.With the scalpel, starting from the nuchal crest, the levator auris muscle was gently dissected away from the occipital crest.The muscle was further separated from the crest using a periodontal elevator, taking care not to shred or split the muscle body.The muscle and skin were retracted to expose the cisternal membrane.A 22G needle with the bevel bent to 45 • was used to make a small incision (1 mm) through the dura, whereon cerebral spinal fluid began to pulse from the incision.The prepared catheter was steadily fed down the intrathecal space to a length of 8.5 cm, watching for any muscle twitching or spasms.If twitching or spasms were observed, the catheter was retracted, redirected, and refed until it could be fed without issue.
Upon placement, the catheter was flushed with 10 μL NaCl 0.9% and again observed for any signs of twitching or spasms.If twitching, spasms, or retraction of an extremity were observed during the flush, the catheter was removed and re-fed until free of deleterious effects.The catheter was externalized through the skin of the skull, between the ears.A metal plug was fitted, and the incision closed in a single layer, using one of the skin sutures to secure the muscle in place.During that same anesthetic session, each animal was given IN MBP 84-104 (above).At day 7 after intrathecal catheter implant and IN, animals received a single IT injection of LXR agonist, GW3695 (6.2 μg/10 μl), or vehicle (1% DMSO and 4% Tween-80 in PBS), as published (Xu et al., 2017).

von Frey testing
Sensitivity to non-noxious mechanical stimuli was measured using a von Frey test.Rats were habituated to the testing environment prior to baseline tests and randomly assigned to experimental groups.Testing was performed during the light cycle by an experimenter blinded to the treatment groups.Rats were placed in individual Plexiglas compartments with a wire mesh bottom.The plantar surface of the hind paw within the sciatic nerve innervation area was stimulated using calibrated von Frey filaments (Stoelting, Wood Dale, IL, USA).Stimuli were applied for 4-6 s with a 0.4-15.0g buckling force to the mid paw plantar surface with ascending filament stiffness until a paw withdrawal response occurred.Stimuli were separated by several-second intervals or until the animal was calm with both hind paws placed on the grid.The consecutive way of applying filaments was continued until six responses were recorded.The 50% threshold was calculated using Dixon's up-down method (Chaplan et al., 1994).

Intracellular calcium imaging
Dissociated DRG cells were loaded with the ratio metric Ca 2+ indicator dye Fura-2-acetoxymethyl ester (2 μM; Molecular Probes) for at least 40 min at 37 • C in 10 mM HEPES (pH 7.4) containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , and 11 mM glucose (extracellular solution).The cells were then transferred to a recording chamber placed on a microscope (Nikon Eclipse) and continuously perfused with the oxygenated (95% O 2 and 5% CO 2 ) extracellular solution (2 ml/min) at ambient temperature.The intracellular calcium concentration was expressed as the 340/380 ratio.The signals were captured and analyzed using the NIS-Elements AR software program (Nikon).All chemicals were directly applied to the bath.The MBP 84-104 or SCR peptide (5 or 10 μg/ml, each) was administered to female and male DRG.Where indicated, capsaicin (500 nM) was co-administered.Calcium imaging (ratio of 340/380) was used to record the DRG responses to the MBP 84-104 or SCR peptide and capsaicin.

LC-MS/mass spectrometry of MBP 84-104 pull-down
Unless indicated otherwise, all procedures were performed at 4 • C. Nerve tissues were washed with PBS and then solubilized for 1 h using 50 mM Octyl-β-d-glucopyranoside (Octyl) in the pull-down buffer (50 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 ).Insoluble material was removed by centrifugation (14,000×g; 20 min).Normalization was done by weighing the tissue and protein concentration determined using the Coomassie Protein Assay kit.Lysate aliquots (500 μg total protein; 2 ml, each) were 2-fold diluted using the detergent-free pull-down buffer to reach a 25 mM final concentration of the detergent.To remove the non-specific binders, the samples were precleared for 4 h using the biotin-labeled SCR immobilized on Streptavidin-beads (200 μl, 50% slurry).The fall-through fraction was co-incubated for 16-18 h with the biotin-labeled MBP 84-104 immobilized on Streptavidin-beads.After extensive washing with 25 mM Octyl in pull-down buffer, the bound material was eluted from the individual WT-and SCR peptide immobilized-beads using 2x reducing SDS-loading buffer.The eluted samples were separated in a 4-12% NuPAGE MOPS gel followed by silver staining.Four individual gel sections discriminating the WT sample from the SCR sample were subjected to in-gel reduction (50 mM DTT, 60 • C, 60 min), alkylation (50 mM iodoacetamide, 40 • C, 45 min in the dark), and digestion using Sequencing Grade Modified Trypsin (Promega; 25 μg/ml, 37 • C).Peptides were analyzed by LC-MS/MS using a Proxeon EASY nanoLC system (Thermo Scientific) coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific).Peptides were separated using an analytical C 18 Acclaim PepMap column 0.075 × 500 mm, 2 μm particles (Thermo Scientific).The mass spectrometer was operated in positive data-dependent acquisition mode.MS1 spectra were measured with a resolution of 60,000, an AGC target of 1 × 10 6 and a mass range from 350 to 1400 m/z.Up to 10 MS2 spectra per duty cycle were triggered, fragmented by collision-induced dissociation, and acquired in the ion trap with an AGC target of 1 × 10 4 , an isolation window of 2.0 m/z and a normalized collision energy of 35.Mass spectra were analyzed using MaxQuant software version 1.5.5.1.MS/MS spectra were searched against the Rattus norvegicus Uniprot protein sequence and the GPM cRAP sequence database of commonly known protein contaminants.Carbamidomethylation of cysteines was searched as a fixed modification, while oxidation of methionines and acetylation of protein N-terminal were searched as variable modifications.Enzyme was set to trypsin in a specific mode and a maximum of two missed cleavages was allowed for searching.The target-decoy-based false discovery rate filter for spectrum and protein identification was set to 1%.

Statistical analyses
Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) by one-way or two-way analyses of variance (ANOVA) for repeated measures for comparing three or more groups, followed by the post hoc Sidak's, Bonferroni, Tukey-Kramer's or Mann Whitney rank sum tests, as indicated in Figure Legends.For parametric statistics, the data was confirmed to be normally distributed first.A two-tailed, unpaired Student's t-test was used for comparing two groups.p ≤ 0.05 was considered significant.
To conduct in-depth assessment of this finding, we hypothesized that upon IN injection in sciatic nerve, MBP 84-104 peptide interacts and affects the function of one or more components of the LXR/RXR complex (Fig. 1C) at one or more sites ipsilateral to the damaged and/or IN injected neuraxis.The axis represents a complex of an LXR with its obligate partner RXRα and a co-activator (CoA).Upon stimulation with an oxysterol ligand (L) such as 25-OHC or a cholesterol precursor (P) such as desmosterol, LXR binds to LXR response element (LXRE) on a promoter of a target gene including Abc cholesterol transporter or IL-6, leading, respectively, to induction or suppression of their transcription and expression.
The changes in LXR, RXR and target genes controlled by MBP 84-104 were assessed using targeted transcriptome analyses of our published RNA-seq dataset (GEO GSE107159) (Chernov et al., 2020).The dataset was obtained at day 7 after IN MBP 84-104 or SCR peptides (30 μg in 3 μl in PBS, each) or PBS (3 μl) into an intact sciatic nerve.Heatmaps of gene expression (absolute values) are shown in the nerve injection site (Fig. 1D) and the ipsilateral DRG and spinal cord (dorsal quarter, Suppl.Fig. S1).IN MBP 84-104 caused specific (not observed in SCR or PBS groups) downregulation of LXRα (encoded by Nr1h3), its obligate partner, RXRα (encoded Rxra), and bile acid farnesoid X receptor (FXR, encoded by Nr1h4) genes in the nerve of both sexes (Fig. 1D).LXRβ (encoded by Nr1h2) and other RXR genes were expressed constitutively and unaffected by any treatment.IN MBP 84-104 induced Cacna2d1 gene in nerves of both sexes (Fig. 1D).With the exception of FXR/Nr1h4, whose expression was restricted to the nerve, all Nr1h and Rxr genes were constitutively expressed in DRG and spinal cord and unaffected by any treatment (Suppl.Fig. S1).At the injection site, IN MBP 84-104 regulated transcription of LXR target genes, Abcg4 cholesterol transporter, apolipoproteins Apoc2, Apoh and Apoa2, and cholesterol hydroxylase CH25H responsible for 25-OHC synthesis (Suppl.Fig. S1).
To further elucidate the role of the LXRα/RXRα axis in femalespecific pronociceptive MBP 84-104 action, we used the model of rat sciatic nerve CCI of both sexes to analyze the changes in LXRα receptor (Fig. 2), its ligands (Fig. 3), and to identify the related MBP 84-104 interactors (Fig. 4).We then tested the effect of LXR stimulation on MBP 84-104 -and estradiol-induced transcriptional activity in sensory neurons in vitro (Fig. 5) and IN MBP 84-104 -induced pain in vivo (Fig. 6).

Cholesterol-related LXRα ligand and receptor in sciatic nerve post-CCI
LXRα (50 kDa) immunoblotting was conducted at days 0 (normal), 7 and 27 post-CCI in rat sciatic nerve (injury site, n = 6/group, 3 male and 3 female).GAPDH (36 kDa) was used as a normalizer (Fig. 2A-B).In nerve whole lysates, LXRα was constitutively expressed and significantly elevated at days 7 and 27 post-CCI with no apparent difference between the sexes.To analyze for potential membrane translocation of nuclear LXR (Ishikawa et al., 2013), the nerves were separated into 12 sucrose density gradient membrane fractions: light/buoyant fractions 4-6 defined as lipid rafts (marked by flotillin-1, 48 kDa), and heavy/non-buoyant fractions 10-12.In the normal and CCI (day 7) nerves, LXRα localized exclusively to the heavy non-lipid raft fractions regardless of sex (Fig. 2C, female shown).In sciatic nerve of both sexes, LXRα is produced by Schwann cells in vivo (Fig. 2D, normal female nerve shown) and in vitro (Fig. 2E).Other LXRα-reactive cells in nerve were endothelial cells and CD68-reactive macrophages (not shown).LXRα levels or distribution showed no statistically significant sex difference in normal or CCI nerve at all time-points.
Sciatic nerve lipidomics was used to assess sex-and injury-dependent changes in cholesterol precursor and metabolite LXRα ligands (Fig. 3) in rat sciatic nerves at days 0 (sham) and 3 post-CCI.Two LXRα ligand intermediate precursors in the cholesterol biosynthetic pathway, desmosterol (Fig. 3A) and 7-dehydrocholesterol (DHC, Fig. 3B), reduced post-CCI.Both were elevated in female relative to male nerves at baseline and post-injury.The cholesterol levels followed the patterns of the precursors, but the finding was not statistically significant (Fig. 3C).The levels of 25-OHC, the key oxysterol LXRα ligand, increased post-CCI in nerves of both sexes, with levels significantly elevated in females relative to males (Fig. 3D).Other sterol profiles displayed injury-dependent changes of interest for future in-depth study (Suppl.Fig. S2).

Ncoa1 as an interactor of MBP 84-104 in sciatic nerve post-CCI
To gain mechanistic insight into the pronociceptive IN MBP 84-104 action, we set to identify its interactors using LC-MS/MS analysis in the rat sciatic nerve at days 0 (sham) and 3 post-CCI (Fig. 4A).The nerves were lysed, and the non-specific binders were pre-cleared on the biotinlabeled SCR peptide immobilized on Streptavidin-beads.The fallthrough material was allowed to bind to biotin-labeled MBP 84-104 immobilized on Streptavidin-beads.The proteins bound to SCR and MBP 84-104 columns were eluted, separated by electrophoresis followed by silver staining.The four protein bands distinct in the MBP 84-104 relative to the SCR sample were excised, digested with trypsin and the tryptic peptides were analyzed by LC-MS/MS.
Because endogenous MBP 84-104 release is observed in the injured only nerve (Chernov et al., 2018;Hong et al., 2017;Kobayashi et al., 2008;Lee et al., 2022;Liu et al., 2012;Remacle et al., 2018), we were especially interested in Ncoa1 and its co-localization with the degraded MBP (dMBP) in nerve at day 3 post-CCI.Ncoa1 and dMBP co-distributed within the same myelinated Schwann cell-axon units (Fig. 4B).Clusters of Ncoa1+ cells non-reactive for dMBP were also noted.These data support a model whereby an injury-specific release of MBP 84-104 may limit the ability of Ncoa1 to activate the LXR/RXR axis (Fig. 4C).This finding alone does not explain the sexual dimorphism of MBP 84-104 action.
Our findings thus far suggest that sex differences in LXR/RXR action may arise from LXRα ligand, not receptor or co-activator, levels.To test whether LXRα ligand stimulation differentially controls pronociception signaling, we next employed synthetic LXR agonist GW3965 in treatment of cultured DRGs of both sexes.
Expression of Erα (ESR1), Erβ (ESR2), LXRα (Nr1h3), and LXRβ (Nr1h2) was confirmed using Taqman RT-qPCR, immunofluorescence and/or immunoblotting and showed to be comparable in male and female DRG cultures (Suppl.Figs.3-5) and not reactive to MBP 84-104 or SCR treatment regardless of sex (p > 0.05, Suppl.Fig. 5A).ESR1 (Suppl.Fig. 3) and LXRα (Suppl.Fig. 4) nuclear distribution was observed in neurons identified by NeuN (neuron marker) and NF200 (large-diameter neuron marker) in vivo and in vitro.ESR1 and/or LXRα reactivity in GFAP-positive glia and CD68-positive macrophages was noted.No apparent sex difference in the expression levels or distribution of either nuclear receptor was observed before or after CCI.
The female and male DRG cultures were treated with 17β-estradiol (100 nM, the ESR1/ESR2 agonist) or PPT (100 nM, an ESR1-selective agonist) in the presence of GW3965 (1 μM, LXRα/β agonist) or DMSO vehicle, followed by Taqman qRT-PCR for LXR-downstream transporter Abca1, Abcg1, and pronociceptive Il6 and Cacna2d1 gene expression (Fig. 5), selected as the factors shown to mediate IN MBP 84-104 induced pain (Chernov et al., 2018;Ko et al., 2016).In a subset of cultures, 17β-estradiol or PPT treatment was done in the presence of the MBP 84-104 peptide (10 μg/ml).GW3965 treatment was administered 24 h after the estrogens and/or MBP 84-104 .In DRG cultures of both sexes, 17β-estradiol produced a highly significant (p > 0.0001) 3-fold induction of Il6 (Fig. 5A) and Cacna2d1 (Fig. 5B) expression.The effect of the PPT to induce Il6 but not Cacna2d1 mRNA was statistically significant.GW3965 treatment attenuated the increase in Il6 and Cacna2d1 caused by the estrogens.GW3965, but not other, treatments induced Abca1 (Fig. 5C) and Abcg1 (Fig. 5D) expression in DRG cultured of both sexes.MBP 84-104 or SCR peptides produced no significant effect on the expression of either gene.
The effect of MBP 84-104 treatment on evoked Ca 2+ transients in rat male and female DRG neurons was assessed using intracellular calcium imaging after MBP 84-104 (5 and 10 μg/ml) or SCR (10 μg/ml) peptide application for 10 min.Responsiveness to the algogenic compound capsaicin (500 nM) was used for control.The capsaicin sensitivity is evident by the strong calcium signals in the TRPV1-positive DRG neurons of both sexes (Suppl.Fig. 5B).Both MBP 84-104 and SCR elicited no effect in neurons of both sexes.Due to no observed impact on TRPV1positive or TRPV1-negative neurons, the changes in the 340/380
We have previously demonstrated that IN MBP 84-104 induced femalespecific allodynia and have attributed it to a 3-tier process of pronociceptive transcriptional remodeling (Chernov et al., 2020).Comparable at the nerve injection site of both sexes, it progressed to the segmental DRG and spinal cord selectively in females.The aim of the present study was to elucidate the role of sex-dependent changes in the nerve that progressed into the DRG and/or spinal cord, including female-specific cholesterol accumulation due to reduced Cholesterol Efflux, increased Cholesterol Biosynthesis signaling, as well as impaired LXR/RXR signaling and its downstream IL-6 in females (Chernov et al., 2020;Ko et al., 2016;Liu et al., 2012).
Cholesterol homeostasis in peripheral nerve is controlled by the heirarchial transcription factor network that includes LXRs and RXRs (Bookout et al., 2006).IN MBP 84-104 initiated a manifold control of nerve cholesterol homeostasis.First, IN MBP 84-104 repressed expression of LXRα and RXRα at the nerve injection site of both sexes.This finding was not recapitulated in the damaged nerves, where LXRα levels were elevated in both sexes.This discrepancy can be potentially explained by high contribution of LXRα-expressing macrophages in the injured nerve, that are not recruited in the nerve post-IN MBP 84-104 (Liu et al., 2012).
MBP 84-104 may regulate the synthesis of cholesterol precursor and oxysterol LXRα ligands.Thus, IN MBP 84-104 induced the expression of 7dehydrocholesterol reductase (DHCR7), which converts cholesterol precursor 7-DHC to cholesterol, particularly, in female nerves (Chernov et al., 2020).Endogenous MBP 84-104 release may contribute to prevalence of 7-DHC in injured nerve of females relative to males.Similarly, the injury-induced increase in 25-OHC oxysterol in female correlates to the ability of MBP 84-104 to induce cholesterol hydroxylase CH25H involved in 25-OHC synthesis (Chernov et al., 2020).
Further, the nuclear receptor co-activator Ncoa1, also known as Src1, emerged as an injury-specific interactor of MBP 84-104 in nerve of both sexes.By interacting with ligand-bound LXRs and RXRs, Ncoa1 represses expression of inflammatory genes and induces expression of antiinflammatory genes (Cermenati et al., 2010).Ncoa1 serves to activate a large number of ligand-controlled nuclear receptors, including DNA-bound ESR1 stabilized by estrogens (Glass and Ogawa, 2006) and IN MBP 84-104 -controlled FXR, vitamin D receptor (VDR), peroxisome proliferator-activated receptor (PPAR) (Chernov et al., 2020;Ko et al., 2016;Liu et al., 2012).Thus, through the relationship with Ncoa1, MBP 84-104 likely controls activities of several nuclear receptors of the hierarchical transcription factor network, including sex steroid receptors.
No sexual dimorphism was observed in MBP 84-104 release (Lee et al., 2022), interaction with Ncoa1 or regulation of LXR post-CCI.Sex-and injury-dependent changes in LXR ligands likely determine sex specificity and degree of anti-nociceptive LXR/RXR action.In support of this assumption, treatment using a synthetic LXR ligand agonist GW3965 attenuated IN MBP 84-104 -induced mechanical allodynia in females at the dose and route of delivery shown efficacious in a male model of peripheral nerve trauma (Xu et al., 2017).IT delivery of GW3965 is expected to activate both LXRs constitutively expressed in DRG and spinal cord.Yet activity of LXRα, not LXRβ, is believed to prevent the development of mechanical allodynia.Anti-nociceptive activity of LXR stimulation limits ER-stress and fosters cholesterol efflux in DRG neurons (Bao et al., 2017;Cermenati et al., 2010;Gavini et al., 2018;Li et al., 2019;Xu et al., 2017).
Based on the present findings, we propose a model whereby LXR/ RXR induces Abc cholesterol transporter and represses estrogen/ESR1induced Cacna2d1 and IL-6 gene transcription in myelinated Schwann cell-A-afferent neuron units of both sexes.By Ncoa1 sequestration, MBP 84-104 blocks transcriptional LXR/RXR activity thus fostering pronociceptive effects of ESR1.Due to high levels of circulating estrogens in females relative to males, this effect of MBP 84-104 is prevalent in females (Fig. 7).
Conversely, low circulating estrogens in males result in low activity of ESR1 in DRG neurons.The injury-and sex-related differences in cholesterol precursors and metabolites suggest the importance of future studies of sterol lipid metabolism in the somatosensory neuraxis, including local synthesis of estrogens in DRG and spinal cord (Tran et al., 2017) and role of oxysterols in binding and partial inactivation of ESR1 (DuSell et al., 2008).Importantly, at comparable treatment doses and conditions, LXR stimulation counteracted pronociceptive activity of estrogen/ESR1 in cultured DRGs of both sexes, consistent with the model that LXR and ESR ligand levels determine sex specificity and degree of anti-nociceptive LXR/RXR action.
Ultimately, MBP 84-104 is an immunodominant epitope implicated in autoimmune demyelinating disease (Harauz and Boggs, 2013).IN MBP 84-104 allodynia is mediated by ganglionic and/or spinal IL-6 (Ko et al., 2016) and depends on adaptive immune activity.Thus, T cell activation is comparable in nerves of both sexes exposed to IN MBP 84-104 ; while localized to nerves in males, T cells are recruited into DRG and spinal cord exclusively females (Chernov et al., 2020).T cell-deficient athymic nude female rats fail to develop allodynia after IN MBP 84-104 (Liu et al., 2012).
The endogenous MBP 84-104 epitope release in CCI nerves of both sexes leads to female-specific circulation of anti-MBP 84-104 IgM autoantibodies, indicating sex-dependent B cell action (Lee et al., 2022).Although degenerative changes in nerves post-IN MBP 84-104 are absent and expression of major proinflammatory cytokines is unchanged relative to the scrambled peptide (Chernov et al., 2018(Chernov et al., , 2020;;Hong et al., 2017;Ko et al., 2016;Liu et al., 2012), the role of innate immune activity in IN MBP 84-104 -induced allodynia cannot be ruled out.This immune cell-and myelin-dependent (Liu et al., 2012;Shubayev et al., 2016) pronociceptive action of IN MBP 84-104 in vivo was not recapitulated in the dissociated unmyelinated DRG cultures.
Because the MBP 84-104 sequence is identical in human, mouse and rat, we expect our findings in rodent models to be of relevance to traumatic, autoimmune and other neuropathies in humans that feature a targeted MBP 84-104 release.Thus, using the same assay we identified anti-MBP 84-104 autoantibodies in female rats post-CCI and in women with multiple sclerosis pain, fibromyalgia (Remacle et al., 2018) and low back radiculopathy (Schuster et al., 2022).Due to its structural homology with acetylcholine M2 receptor and a human coronavirus CoV-OC43 p65-like protein, we argue that MBP 84-104 contributes to complex regional pain syndrome, viral and idiopathic neuropathies (Ko et al., 2016;Liu et al., 2012;Shubayev et al., 2016Shubayev et al., , 2018Shubayev et al., , 2022)).
In conclusion, bioactive MBP fragments released in the damaged PNS regulate cholesterol and sex steroid metabolisms by interaction with a nuclear receptor coactivator, Ncoa1.Restoring LXR/RXR activity presents a therapeutic strategy in management of neuropathic pain induced by bioactive MBP.Elevated levels of cholesterol precursors in female relative to male suggest a future study of fundamental sexual dimorphism in sterol composition of peripheral nerves.
Fig. 1.MBP 84-104 suppresses LXRα/RXRα axis in sciatic nerve.A, The degraded (d)MBP fragment comprising the MBP 84-104 epitope is released in nerve between days 3 and 27 post-CCI(Lee et al., 2022;Liu et al., 2012) (a schematic).B, Sequence alignment of the MBP central domain showing strong sequence conservation with 100% identity between human and rodent MBP 84-104 regions.C, The LXR/RXR axis (a schematic).Upon activation by cholesterol precursor (P) or oxysterol ligand (L), LXR dimerizes with RXR, binds to LXR-responsive-element (LXRE) on a target gene promoter leading to induction (red arrow) of Abc cholesterol transporter or suppression (blue arrow) of IL-6 expression.D, Heat maps of normalized absolute gene expression values (Log2TPMs, transcripts per million reads) for RNA-seq dataset (GEO ID GSE107159) obtained in sciatic nerve (injection site) after IN MBP or SCR peptide or PBS in mice in n=12/group (6 male, 6 female(Chernov et al., 2020).Gene subsets were selected based on Gene Ontology annotations for LXR/Nr1h, RXR/Rxr and Cacna2d1 genes.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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caption on next page) S.K. Hullugundi et al. ratios were not recordable.We conclude that DRG neurons of both sexes: (a) express ESRs and LXRs; (b) induce Il6 and Cacna2d1 genes upon 17β-estradiol activation via ESR1; (c) block the 17β-estradiol/ESR1 action upon LXR stimulation; (d) induce cholesterol transporter genes upon LXR stimulation; (e) do not change gene expression or intracellular calcium concentration in response to MBP 84-104 .